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A three-domain copper-nitrite reductase with a unique sensing loop

DOI: 10.1107/S2052252519000241 DOI Help

Authors: Diederik Johannes Opperman (University of the Free State) , Daniel Horacio Murgida (Universidad de Buenos Aires) , Sergio Daniel Dalosto (CONICET-UNL) , Carlos Dante Brondino (Universidad Nacional del Litoral (UNL), CONICET, Ciudad Universitaria) , Felix Martín Ferroni (Universidad Nacional del Litoral (UNL), CONICET, Ciudad Universitaria)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Iucrj , VOL 6

State: Published (Approved)
Published: March 2019
Diamond Proposal Number(s): 15292

Open Access Open Access

Abstract: Dissimilatory nitrite reductases are key enzymes in the denitrification pathway, reducing nitrite and leading to the production of gaseous products (NO, N2O and N2). The reaction is catalysed either by a Cu-containing nitrite reductase (NirK) or by a cytochrome cd1 nitrite reductase (NirS), as the simultaneous presence of the two enzymes has never been detected in the same microorganism. The thermophilic bacterium Thermus scotoductus SA-01 is an exception to this rule, harbouring both genes within a denitrification cluster, which encodes for an atypical NirK. The crystal structure of TsNirK has been determined at 1.63 Å resolution. TsNirK is a homotrimer with subunits of 451 residues that contain three copper atoms each. The N-terminal region possesses a type 2 Cu (T2Cu) and a type 1 Cu (T1CuN) while the C-terminus contains an extra type 1 Cu (T1CuC) bound within a cupredoxin motif. T1CuN shows an unusual Cu atom coordination (His2–Cys–Gln) compared with T1Cu observed in NirKs reported so far (His2–Cys–Met). T1CuC is buried at ∼5 Å from the molecular surface and located ∼14.1 Å away from T1CuN; T1CuN and T2Cu are ∼12.6 Å apart. All these distances are compatible with an electron-transfer process T1CuC → T1CuN → T2Cu. T1CuN and T2Cu are connected by a typical Cys–His bridge and an unexpected sensing loop which harbours a SerCAT residue close to T2Cu, suggesting an alternative nitrite-reduction mechanism in these enzymes. Biophysicochemical and functional features of TsNirK are discussed on the basis of X-ray crystallography, electron paramagnetic resonance, resonance Raman and kinetic experiments.

Keywords: Thermus scotoductus SA-01; three-domain copper-nitrite reductase; X-ray crystal structure; SerCAT residue; sensing loop

Subject Areas: Biology and Bio-materials

Beamlines: I04-1-Macromolecular Crystallography (fixed wavelength)